X-ray crystal structures have been used to assemble a model of an intact multisubunit tethering complex of the COG/Dsl1/exocyst/GARP family (Koumandou et al., 2007
). The model reveals a tower 20 nm in height with a disordered lasso at its tip. This structure appears to be highly consistent with the inferred role of the Dsl1 complex in COPI vesicle capture (). At its base, the Dsl1 complex is anchored to the ER membrane via a bivalent attachment to two different ER SNAREs, Use1 and Sec20. Consistent with our biochemical analyses, co-immunoprecipitation experiments demonstrate that near-stoichiometric quantities of Use1 and Sec20 are recovered in association with the Dsl1 complex (Kraynack et al., 2005
). The lasso, on the other hand, contains Trp-containing sequence motifs capable of binding subunits of the coatomer (Andag et al., 2001
; Andag and Schmitt, 2003
; Zink et al., 2009
). The position of these motifs at the distal tip of the complex, within a long (111 residues in S. cerevisiae
) disordered segment capable of extending 10 nm or more, seems ideally suited to the purpose of capturing COPI vesicles residing within a wide sweep of three-dimensional space. It has recently been proposed that binding of the vesicle to the Dsl1 complex impacts vesicle uncoating (Zink et al., 2009
). Our results imply that tethering, uncoating, SNARE assembly, and membrane fusion may be coordinated by the Dsl1 complex. Our structural model, moreover, provides a foundation for investigating this hypothesis further.
The Dsl1 tower is not monolithic; instead, electron microscopy of negatively stained samples, in conjunction with image classification, indicates that it contains a flexible hinge at the center of the Dsl1 subunit. The linkage between the Dsl1 and Tip20 subunits, mediated by an antiparallel association between N-terminal helices, is probably flexible as well (Tripathi et al., 2009
). Thus, although in our structural model the SNARE binding regions of Sec39 and Tip20 are relatively close to one another, the flexible hinge(s) would allow spatial separation of these subunits. The flexibility and/or conformation of one or more of the hinges might be linked to the succession of events that represent a functional cycle. It is striking in this regard that the lasso lies in close proximity to the Dsl1 subunit’s central hinge, suggesting that engagement of the lasso by a COPI vesicle binding could influence this hinge and, in turn, control the relative positioning of Sec39 and Tip20 ().
The Dsl1 complex, in addition to binding individual ER SNAREs via their N-terminal regulatory domains, also binds assembled SNARE complexes. In this way, it appears capable of remaining associated with ER SNAREs throughout their functional cycle, as depicted in . Moreover, the Dsl1 complex accelerates SNARE complex assembly in vitro. This acceleration, though quantitatively modest, is specific inasmuch as it depends on the integrity of the MTC. One potential mechanism by which an MTC might regulate SNARE assembly is by altering the conformations of the individual SNAREs. This mechanism seems attractive based on previous studies of SNARE assembly, which indicated that regulatory domains in some SNAREs are capable of folding back upon, and protecting from assembly, the SNARE motifs (Munson et al., 2000
; Tochio et al., 2001
). Tethering factors that engaged these regulatory domains might thereby deprotect the SNARE motifs, accelerating assembly. The addition of Sec39 alone did, in fact, appear to accelerate SNARE assembly (Figure S5
). In a related MTC, the opposite result was obtained: an individual exocyst subunit, Sec6, was found to bind the SNARE protein Sec9 and thereby to inhibit the assembly of exocytic SNAREs (Sivaram et al., 2005
). Clearly, the regulation of SNARE conformation and assembly by individual MTC subunits remains an open question deserving further exploration.
A second, and perhaps the simplest, mechanism by which the Dsl1 complex might influence SNARE assembly is by serving as a scaffold, binding simultaneously to two of the four SNAREs needed to form a stable complex, and possibly helping to orient them for productive interaction (). This mechanism demands that the Dsl1 complex be intact, as suggested by our results, since it uses different subunits to bind different SNAREs. Moreover, the flexibility inherent in the Dsl1 complex might be important, although a priori it is not obvious whether it would assist or impede assembly. It does seem clear, however, that additional factors that affected the conformation and/or flexibility of the Dsl1 complex could greatly enhance (or inhibit) its assembly activity.
In conclusion, we present here a structural model for an intact MTC of the COG/Dsl1/exocyst/GARP family. Two of its three subunits (Dsl1 and Tip20) are homologous to previously reported structures of individual COG and exocyst subunit fragments, while one (Sec39) displays a novel fold. We demonstrate that the integrity of the Dsl1 complex is important both for its function in vivo and for its ability to accelerate SNARE assembly in vitro. Our analysis also reveals what we believe to be the first direct evidence for structural flexibility within an MTC. This flexibility takes two forms. First, a mobile lasso for vesicle capture is presented at the top of the Dsl1 tower. Second, hinge motions within the body of the tower appear to allow for conformational changes during a functional cycle. It will be particularly interesting, going forward, to characterize these conformations, their modulation by other factors, and their specific roles in orchestrating vesicle capture and fusion.